Abstract
Background & Objectives
Wound healing is the complex physiological process of replacing damaged cells or tissue layers. The neem (Azadirachta Indica) has a variety of biological activities, which may hasten the rate at which the wound healing mechanism occurs. Silk fibroin is a biomaterial that is reported for its tissue regeneration activity. So, the present study was designed to assess the effectiveness of a hydrogel comprising neem and silk fibroin biomaterials for the treatment of wounds.
Methods
Topical neem hydrogels (N-HG) with and without silk fibroin (N-SFB-HG) were prepared using neem extract, silk fibroin, and guar gum, which act by entrapping the components by forming a gel. Evaluation tests such as Fourier transform infrared spectroscopy (FT-IR), visual emergence, pH, rheological behavior, spreading capacity, drug content, skin irritation, anti-microbial action, in vivo wound healing activity, and stability were carried out.
Results
The FT-IR results showed no chemical interaction between the constituents. The formed hydrogels had pH values of 5.87 ± 0.3 for N-HG and 5.76 ± 0.2 for N-SFB-HG. The preferred topical gel viscosity was observed in the N-HG (54.2 ± 3.2cPs) and N-SFB-HG (59.9 ± 4.8cPs) formulations. The formulated hydrogels were sterile and did not irritate the skin. The in vivo wound healing investigation results reveal that the N-SF-HG treatment speeds up the regeneration of the injured area faster when compared to control and N-HG treated groups.
Interpretation & Conclusion
These results support the efficacy of the topical hydrogel formulation, including neem and silk fibroin. Therefore, the neem-silk fibroin hydrogel formulation is a therapeutically viable choice that, following necessary clinical research, might be utilized in novel formulations for managing chronic wounds.
Keywords: Azadirachta Indica, Neem, Hydrogel, Wound healing, Silk fibroin
1. Introduction
Wound healing is a natural regenerative mechanism where the replacement of damaged tissue occurs. During the initial stages of inflammation, attempts are made to heal the lesion brought on by local damage. In the end, they lead to a repair that includes collagen deposition and regeneration, which triggers the process of cell proliferation (Shaw and Martin, 2009, Gowda et al., 2023). External, internal, physical, chemical, electrical, or thermal stimulation can result in lesions. Though it looks simple, it emerged as a challenge to the field of research (Kumar et al., 2005, Gonzalez et al., 2016). The primary purpose of wound management is to repair the wound at the earliest possible, with less discomfort, pain, and scarring to the patient. Hence, many types of research are ongoing to meet the needs of the current situation (Singer and Clark, 1999, Hees, 2012, Diegelmann and Evans, 2004, MacKay and Miller, 2003). Neem (Azadirachta Indica) is a widely accepted medicinal plant with a large spectrum of biological activity. In Ayurveda and homeopathy, it is a common home remedy for a number of diseases. The anti-bacterial, anti-inflammatory, anti-fungal, and anti-viral characteristics of neem's active components, such as nimbidin, nimbin, and nimbidol, aid in promoting the healing of wounds. Moreover, neem has a high concentration of minerals, vitamins, and amino acids crucial for wound healing during the proliferation phase (Subapriya et al., 2005). Silk is a naturally occurring protein that is extracted from mulberry silk (Nasrine et al., 2022). The exterior protein sericin, which may trigger an immune reaction when combined with fibroin, is removed (Altman et al., 2003). Outstanding biocompatibility, excellent ability to get disintegrated, incredible mechanical strength, and minimal immunogenic capabilities are all characteristics of silk fibroin (SFB). As a result, it has been extensively employed in applications for tissue engineering and regenerative medicine (Narayana et al., 2023, Sixma and Van Den Berg, 1984, McDonald et al., 2007). Its application in skin regeneration processes, hemostatic qualities, low inflammatory potential, permeability to oxygen, and water vapor have all been well explored (Minoura et al., 1990, Santin et al., 1999, Lu et al., 2015). In small animal models, it has been documented that SFB dressing stimulates quicker wound recovery and more remarkable skin reconstruction when compared to hydro active dressings. Also, it was determined to be safe in tests for skin sensitivity, acute cutaneous toxicity, and acute dermal discomfort (Padol et al., 2013). Various methods are used to manage pain, inflammation, and skin conditions, sanitizing skin, and acting as sustained release tools in wound healing. Numerous research groups formulated transdermal hydrogels of anti-inflammatory agents using several polymers (Chittodiya et al., 2013). It was reported that the gels are promising perspectives in wound recovery (Bhowmik et al., 2012, Sanjana et al., 2022). Hydrogel is a physical barrier and provides moisture, promoting wound healing (Narayana et al., 2022). Furthermore, it also fills irregularly shaped wounds and deals with deep bleeding. Therefore, this study aimed to design and evaluate a neem extract-loaded SFB hydrogel formulation to enhance wound healing.
2. Methods
2.1. Materials
Bangalore-based Sericare company provided a complimentary sample of SFB. From Loba Chemicals in Mumbai, guar gum was acquired. None of the other compounds were further purified; they were all analytical reagent grades.
2.2. Preparation of neem extract
Neem leaves were collected from the Yenepoya campus and kept for drying in the shade. 100 g of powders were obtained after grinding the dried leaves. 100 g/1000 ml of distilled water was used to extract the resultant powders for 7 h with continuous shaking at 120 rpm in the orbital shaker apparatus (KEMI, Labon instruments, India, KORB.1 MS) (Fig. 1). The material was filtrated using Whatman No.1 (Whatman, UK) paper, lyophilized, then stored in amber color flasks. To produce the aqueous leaf extract, 1 g of the lyophilized extract was diluted in 100 ml of purified water (0.01 g/ml) (Polaquini et al., 2006).To extract the active ingredients from neem leaves, a variety of polar, semi-polar, and non-polar-solvents-including-ethyl acetate, ethanol, and water, were utilized. According to published research, antioxidant chemicals can dissolve in semi-polar solvents, while other terpenoid compounds, including azadirachtin, can be extracted using either polar or non-polar solvents (Septiyani and Wibowo, 2019).
Fig. 1.
Prepared neem leaf extract.
2.3. Total phenol contents
Adding water, 6% sodium carbonate (Na2CO3) and 10% Folin-Ciocalteu reagent (FCR) were prepared. 4 mg of aqueous neem extract was taken and dissolved with 4 ml methanol, and 200 µL resulting sample was taken from the solution and added to another test tube, along with 1.5–2 ml of 10% FCR reagent, and kept settling for 5 min in the darkened place. The resulting solution, 1.5 ml of Na2CO3 in a concentration of 6%, was added and maintained in the dark condition for around 2 h. The absorbance of the sample was then measured at wavelength 760 nm.
2.4. Antioxidant activity assay
In a test tube, 10 ml of methanol was used to dissolve 2 mg of aqueous neem leaf extract. The following concentrations were used for the serial dilution samples, such as 200, 100, 50, 25, and 12.5 mg/ml. All test tubes received 2.5 ml of a 0.004% α-diphenyl-β-picrylhydrazyl DPPH solution, which was then added, gently mixed and left in a dark place for 90 mins. The wavelength used to measure absorbance was 570 nm. The following formula was used to determine the crude extract's concentration of antioxidant activity (Misal et al., 2012).
2.5. Qualitative phytochemicals analysis
2.5.1. Test for identification of alkaloids
Mayer’s test
Neem extract in 2 ml was put into a test tube along with 2–4 droplets of Mayer's reagent solution. An alkaloid in the tested extract is indicated by the precipitate of a greenish tint forming in the sample solution.
Wagner’s test
After adding 2–4 drops of Wagner's reagent into 2 ml measured neem extract, brick-colored precipitation formed, suggesting the existence of alkaloids.
2.5.2. Test for the identification of flavonoids
Alkaline reagent test
About 2 ml neem extract was collected in a test tube, then 2% w/v of sodium hydroxide solution was put in it. A bright yellow hue emerged. The emergence of a colorless solution with the introduction of 2–4 drops of diluted hydrochloric acid suggests the existence of flavonoids in the sample.
2.5.3. Test for the identification of saponins
Foam test
About 2 ml of neem extract was placed in a cylindrical tube. When the extract is agitated for about 15 mins, a 2 cm thick layer of foam appears in the tube, indicating the existence of saponins.
2.5.4. Test for terpenoids
Salkowski’s test
2 ml of concentrated sulfuric acid (H2SO4) and 2 ml of chloroform were mixed with 2 ml of neem leaf extract. The chloroform layer was red, while the acid layer was brilliant greenish-yellow. This attests to the existence of terpenoids.
2.5.5. Test for glycosides
Keller-Kilian test
The test tube was filled with 2 ml of glacial acetic acid and a 2% solution of FeCl3. The resultant solution was added to another test tube with 2 ml of H2SO4. At the interface, a brown ring indicates that cardiac glycosides are present.
2.5.6. Test for polyphenols and tannins
A 2% dilution of FeCl3 in 2 ml of neem extract. Their slight blueish-green or blue-black hue confirms the existence of polyphenols and tannins.
2.5.7. Test for steroids
Crude neem extract was added to 2 ml of chloroform, followed by concentrated H2SO4 sidewise. The formation of red color in the lower chloroform layer confirms the existence of steroids.
2.5.8. Test for the presence of coumarins
The remaining neem extract residues were diluted in hot water, cooled, and split into 2 test tubes. One of the test tubes received 10% (w/v) ammonium oxide, while the second test tube served as the control. The existence of coumarins is confirmed by the fluorescence hue (Shrestha et al., 2015, Khanal, 2021).
2.6. Quantitative test of phytochemicals
2.6.1. Test for flavonoids
A conical flask holding 100 ml of purified water and 5 gm of extract solution was combined, followed by adding 2 ml hydrochloric acid (HCl) solution to boil for 30 mins after being introduced. The resultant mixture was then cooled down before getting filtered by using weighed Whatman filter paper. After complete filtration, the filter paper was left behind when the filtrate was discarded, and it was dried using an oven (Labmatrix Manufacturing LLP, LMOS.1FD) for 30 min at 60 °C. The below formula was used to calculate the weight of flavonoids.
2.6.2. Test for alkaloids
A 5gm sample of neem leaf extract was diluted in 100 ml of 10% acetic acid and agitated for 4 hrs. The obtained solution was subsequently filtrated, and the filtrate was vaporized using a hot plate and magnetic stirrer to 1/4th of its original volume (REMI 2-MLH). Ammonium hydroxide (NH4OH) concentration was applied dropwise in the hopes of precipitating the alkaloid content. After filtering, the material was rinsed using 1% NH4OH and heated for 30 mins in an oven at 60 °C.
2.6.3. Test for terpenoids
Neem extract weighing 10 g was obtained and dissolved in 90 ml of ethanol. The extract was refiltered using a separating funnel after being combined with 10 ml of petroleum ether. The extract was measured once it was fully dried. Total terpenoids present in the extract were measured by using bellow mentioned formula:
2.6.4. Test for saponins
After adding 25 ml of the extract and 100 ml 50% alcohol to a flask with a flat bottom, the mixture was kept boiling for 30 min. The heated solution that was produced was filtered, and 2 gm of charcoal was then added to the filtrate. An equivalent volume of acetone was mixed with the filtrate to precipitate the formed saponins, and also the precipitated saponins were collected. The bellow mentioned formula was used to calculate the weight of saponins(Harborne, 1973, Muhammad and Abubakar, 2016):
2.7. Formulation of neem and neem-combination of SFB topical hydrogels
2.7.1. Neem extract hydrogels (N-HG)
Neem extract is dissolved in 40 ml of deionized water and thoroughly swirled for one hour at 250 revolutions per minute using a digital mechanical stirrer. Finally, to prevent lumps from forming, guar gum powder (GGP) is added in tiny aliquots while constantly stirring at 500–600 rpm. Afterward, 0.1 gm of sodium benzoate was added slowly, stirring the mixture for 30 min (Table 1).
Table 1.
Composition of N-HG and N-SFB-HG formulation.
| Sl No. | Components | Quantity taken |
|
|---|---|---|---|
| N-HG | N-SFB-HG | ||
| 1 | Neem extract | 0.5 gm | 0.5 gm |
| 2 | GGP | 0.5 gm | 0.5 gm |
| 3 | SFB | – | 0.3 gm |
| 4 | Sodium benzoate | 0.1 gm | 0.1 gm |
| 5 | Deionizer water | 40 ml | 40 ml |
2.7.2. Neem SFB hydrogel (N-SFB-HG)
The same above procedures were followed to prepare N-SFB-HG up to the inclusion of GGP. After that, 0.3 gm of SFB was progressively added while constantly stirring. Next, 0.1 gm of sodium benzoate was slowly added into the solution with 30 mins stirring (500–600 rpm) (Table 1) (Al-Hashemi et al., 2016).
2.8. Evaluation parameters
2.8.1. Fourier transform – Infrared (FT-IR) spectroscopy
Neem extract, SF, GGP, and N-SFB-HG formulation were used in FT-IR investigations utilizing an FT-IR spectrophotometer (Shimadzu, Japan). Looking for any alteration in the peaks, IR spectral studies were conducted to investigate the relationship between the drug and pharmacological excipients. The sample was deposited directly beneath the probing, which was firmly attached. The probe was subsequently scanned between 4000 and 500 cm−1 wavenumbers. The gathered IR spectra allowed identifying functional groups at every wavenumber (cm−1) (Sanjana et al., 2022).
2.8.2. Physical appearance
The formulated gels were examined visually for physical appearance and uniformity (Chen, 2016).
2.8.3. pH determination
The hydrogel formulation's pH was evaluated using a digital pH meter instrument (LAB MAN LMPH-10). Before the pH determination, it was calibrated using a standard phosphate buffer solution. A 1 % hydrogel formulation was taken in deionized water, and pH was determined by dipping the electrode (Kumar et al., 2018).
2.8.4. Determination of viscosity
Using a Brookfield Rheometer DV3T and spindle no. 7 at various rpms at room temperature, the viscosity of the hydrogel compositions was assessed (Monica and Gautami, 2014, Aslani et al., 2018).
2.8.5. Spreadability test
On a glass plate that had previously been marked with circles of 2 cm in diameter, 0.5 g of produced hydrogel was spread evenly across the surface, and any grainy hydrogel was noted. 500 gm of weight was let to rest on the top glass plate while the second glass plate was left positioned over the first. When the hydrogels had been spread out in triplicate, the diameter of the circle was measured and computed using the formula below (Al-Suwayeh et al., 2014).
Where d = Diameter of spread gel.
2.8.6. Drug content
By measuring the drug content, analysis was utilized to establish the uniform dispersion of neem extract in gels. Gels in deionized water were properly mixed, then vortexed for 5 min to create a homogenous mixture. Also, the mixes underwent a 20-minute, 1000-rpm centrifugation process. The resulting samples were analyzed for drug content by using UV–Visible spectrophotometry (SHIMADZU, Japan 1900) at 355 nm (Fong Yen et al., 2015).
2.8.7. Test for sterility
An alternate fluid thioglycolate (FT) medium and soya casein digest (SCD) medium were employed to check the sterility of the synthesized neem hydrogel for the presence of aerobic and anaerobic bacteria and fungi. Comparisons between the test sample's sterility and the positive (growth promotion) and negative (control) controls were made (sterility). Bacillus subtilis and Candida albicans species were utilized. Every time, incubation was carried out, and growth was noticed (Singh et al., 2010).
2.8.8. Anti-bacterial activity tests
The neem hydrogel's anti-bacterial activity will be evaluated by adopting the cup-plate diffusion technique using agar media, and doxycycline as the standard. The agar medium was prepared and sterilized for at least 15 mins at 15 lb/sq inch. An incubator was then used to incubate the prepared agar and placed into the control and test petri plates. Once the agar had solidified, a sterilized borer was used to create cups, filled with the testing solution, and left to incubate for 24 h. Zone of inhibition demonstrates Escherichia coli and Staphylococcus aureus sensitivity to prepared samples (Mandal et al., 2012, Anbarasan et al., 2019).
2.8.9. Skin irritation test
Three mature male Wistar albino rats were placed in the cage for skin irritation research. Rats were provided unlimited access to water and standard food. RH (30–––70%), room temperature (22 ± 3 °C), and artificial light (8 am − 8 pm) were all kept at the proper levels. Each Wistar albino rat had a 2 cm2 portion of its neatly trimmed skin shaved before a 1 ml dose of each gel base, N-HG, and N-SFB-HG gel formulations were applied once. The skin response at the application location was subjectively evaluated and graded for every 1, 24, 48, and 72 h, on the 7th, and 10th days. Skin irritability was tested after 10 days of research to determine the safety of the formulations for wound healing activities (Okamoto et al., 2003).
2.8.10. Ex vivo permeation in rat skin
Phosphate buffer solution with pH 7.4 was employed as the receptor medium in vitro diffusion experiments employing Franz diffusion cells. The skin of the removed rat was fastened to the diffusion cell (donor cell). With constant stirring, 100 ml of receptor media and 1 ml of each hydrogel were diluted significantly. The procedure was conducted at a constant 37 ± 10℃. An aliquot of 5 ml was taken at various times up to 8 hrs and replaced with an equivalent amount of fresh diffusion media. Each time the interval’s cumulative percent release was determined (Taş et al., 2004).
2.8.11. In vivo wound healing studies
Wistar albino adult male rats weighing between 150 and 200 g were utilized for the experiment. The institutional animal ethics committee, Yenepoya (Deemed to be University), approved the investigation. The rats were sedated before and throughout the process of creating the wounds using an intraperitoneal ketamine injection (75 mg/kg) and xylazine (8 mg/kg). The dorsal midline area hairs were shaved using a razor blade. Then, a 2 cm diameter was measured and marked. An excision is made around the marked circle (1 cm) using a scalpel. Rats (n = 6) were divided into three groups. Group I: Topical gel base administration served as a negative control similar to the control. Group II: N-HG was applied topically in this treatment group. Group III: After applying N-SFB-HG topically, the treatment was given daily for 21 days, and groups were observed for how their wounds healed throughout the study period. The severity of the wounds was categorized based on the percentage of wound closure, which was calculated using the following formula:
2.8.12. Transmission electron microscopy (TEM) analysis
The morphology of gel compositions was analyzed using a transmission electron microscopy instrument. A JEM-2100 electron microscope; (Model: JEOL, Tokyo, Japan) was employed to examine the formulations. The negative staining TEM method was used briefly, and parafilm was applied to a 50 μL sample of the gel composition. The carbon-coated grid was used to dry the samples, and they were then stained negatively with a phosphotungstic acid aqueous solution. Once the sample had dried at a 100 kV accelerating voltage, it was examined under a microscope at 10-to-100-fold expansion (Nasrine et al., 2022).
2.8.13. Accelerated stability studies
According to International Council on Harmonization (ICH) criteria, stability testing shows the manufactured formulation's quality at various timeframes and humidity levels. The hydrogel compositions were placed in glass vials and tested for three months under accelerated stability conditions at 25 ± 2 °C and 40 ± 2 °C with Relative Humidity (RH) of 75 ± 5%. Throughout three months, samples were gathered regularly every month and examined for changes in pH, spreadability, viscosity, and drug content. Any difference in evaluation parameters was observed (Ahamed et al., 2011).
2.9. Ethical clearance
The study protocol was approved by Institutional Animal Ethical Committee, Yenepoya (Deemed to be University) (YU/IAEC/16/2019). Male Wistar albino rats (specific-pathogen-free grade, 5––7 weeks old, 150 – 200 g) were used in the study. They were accommodated in ventilated cages under controlled conditions (light and dark cycles; 12 h and 25 °C) with rat pellets and water ad libitum. The animals were procured from Liveon Biolabs, Tumakuru, Karnataka. The experiments were performed at Yenepoya Medical College, Department of Pharmacology, Yenepoya (Deemed to be University) as per CPCSEA guidelines.
2.10. Statistical analysis
The statistical analysis of the collected data we performed using GraphPad Prism Software (Ver. 6.01, La Jolla, California, USA), and data were expressed as mean ± standard deviation (mean ± SD). For the multiple group comparison test, Tukey's multiple comparison tests and variance analysis (one-way ANOVA) were used with statistical significance at a confidence level of 95% (p < 0.05).
3. Results
3.1. Determination of phenol content and antioxidant activity
The FCR method was used to determine the presence of total phenol in the aqueous neem leaf extract. The aqueous neem extract contains 0.57 mg of phenol compounds in 100 gm of dry leaf powder. In this study, the antioxidant activity of aqueous neem leaf extract was determined by in vitro models by the DPPH method shown in Fig. 2, Fig. 3.
Fig. 2.
Standard calibration curve for gallic acid (GA).
Fig. 3.
Anti-oxidant activity of aqueous neem leaf extract.
3.2. Phytochemical analysis by qualitative method
The qualitative tests were used to detect the various phytochemicals present in the aqueous extract of neem leaves. The aqueous neem leaf extract tested positive for all tested phytochemicals except coumarins. The existence of alkaloids was confirmed by both Mayer’s and Wagner’s tests. The alkaline reagent test confirmed the presence of flavonoids. The foam formation and reddish-brown color formation at the interface confirmed the existence of saponins and terpenoids. The positive outcome of Salkowski and Keller kellani tests evidenced the presence of glycosides. The qualitative test for the presence of polyphenols and tannins exhibited positive results. Similarly, unlike the other phytochemicals, the presence of steroids was confirmed by green coloration; and the presence of coumarins was found negative. Fig. 4 depicts the images for the results of various qualitative phytochemical tests of aqueous extracts of neem leaf.
Fig. 4.
Qualitative test of phytochemicals for Neem leaf extract.
3.3. Quantitative test of phytochemicals for neem leaf extract
Quantitative screening was done for various phytochemicals; data are displayed in Table 2. The results indicated that neem leaf extracts contain the highest percentage of saponins, followed by terpenoids and the lowest yield of alkaloids.
Table 2.
Quantitative screening data of neem extract.
| Sl. No. | Phytoconstituents | % Yield |
|---|---|---|
| 1 | Alkaloids | 8.43 ± 0.31 |
| 2 | Flavonoids | 9.57 ± 0.45 |
| 3 | Saponins | 14.8 ± 0.16 |
| 4 | Terpenoids | 12.71 ± 0.9 |
3.4. FT-IR analysis
The FT-IR study was performed to assess any incompatibility between the components. The characteristic peaks present in the neem and polymers were enlisted. FT-IR study outcomes revealed no alteration of peaks, and this indicated that components are compatible with each other. The FT-IR spectrum is depicted in Fig. 5, and the functional group's interpretation is shown in Table 3.
Fig. 5.
FT-IR spectrum of neem extract, SFB, GGP, and N-SFB-HG.
Table 3.
Interpreting results of FT-IR spectra.
| Sample | Absorption (cm-1) | Category | Compound class |
|---|---|---|---|
| Neem | 3400––2600 | O–H stretch (Strong, broad) | –COOH- |
| 3300–3200 | Bending vibration of N–H and –OH stretch | Amine | |
| 1700–1600 | C = O stretch (Strong) | –COOH- | |
| 900–700 | C = C bending | Alkene | |
| SFB | 3500––2000 | O–H stretch (Strong broad) | –COOH- |
| 1620–1600 | N–H bend | Amine | |
| 1650–1400 | N-O stretch (Strong) | Nitro compound | |
| 1300–1000 | C-N stretch (Medium) | Amine | |
| 900–700 | C = C bend | Alkene | |
| GGP | 3400––2600 | O–H stretch (Strong, broad) | –COOH- |
| 1700–1500 | C = C stretch (Weak) | Alkene | |
| 1400–1300 | O–H bend (Medium) | Phenol |
3.5. Organoleptic properties, pH, and viscosity analysis
The organoleptic properties of topical formulations showed that N-HG and N-SFB-HG were homogenous with smooth texture. Hydrogels showed pH within an acceptable range. The viscosity of the developed formulations was within the range of 54.2 to 59.9 cPs. The results are displayed in Table 4.
Table 4.
Visual analysis, pH, viscosity, and % drug content analysis data of formulated hydrogels.
| Formulation code | Visual appearance | pH | Viscosity (cPs) | Drug content (%) | Spreadability (mm/min) |
|---|---|---|---|---|---|
| N-HG | Dense, Greenish cloudy | 5.87 ± 0.3 | 54.2 ± 3.2 | 83 ± 4.1 | 28 ± 4.1 |
| N-SFB-HG | Dense, Greenish cloudy | 5.76 ± 0.2 | 59.9 ± 4.8 | 84 ± 2.9 | 29 ± 2.1 |
*All the analysiswere performed in triplicates (n = 3), and outcomes expressed as mean ± SD.
3.6. Spreadability
Good skin spreadability, ease of product removal from the container, and good bioadhesion of the semisolid formulation are all characteristics of the finest semisolid quality product, which is crucial for greater consumer acceptance. It was observed that there was no sandy surface, and gels were easily spread over the glass plate. The diameters observation for the formulations was carried out for one minute, and obtained data is shown in Table 4.
3.7. Drug content
The percentage amount of neem in N-HG and N-SFB-HG was 83 ± 4.1%, and 84 ± 2.9% respectively. The attained findings showed that uniform distribution of neem extract. The neem content was good in both N-HG and N-SFB-HG formulations, with a low standard deviation. The drug content results are displayed in Table 4.
3.8. Test for sterility
The sterility test evidenced no bacterial or fungal growth. It showed that the medium and tested N-HG and N-SFB-HG were sterile. Hence, the N-HG and N-SFB-HG formulations passed the sterility test, as expressed in Table 5. The various studies reported comparable outcomes.
Table 5.
Sterility test.
| Days | 1 |
2 |
3 |
4 |
5 |
6 |
7 |
|||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| FT | SCD | FT | SCD | FT | SCD | FT | SCD | FT | SCD | FT | SCD | FT | SCD | |
| Positive control | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
| Negative control | – | – | – | – | – | – | – | – | – | – | – | – | – | – |
| N-HG | – | – | – | – | – | – | – | – | – | – | – | – | – | – |
| N-SFB-HG | – | – | – | – | – | – | – | – | – | – | – | – | – | – |
(+) = Represents growth of organism, (-) = absence of growth.
3.9. Anti-bacterial activity
To determine the anti-bacterial potential of the prepared N-HG and N-SFB-HG formulations, it was examined against gram + ve (staphylococcus aureus) and gram-ve (Escherichia coli) bacteria. The N-HG and N-SFB-HG formulation exhibited good ZOI against tested bacteria. The ZOI for N-HG was 26 ± 0.7, N-SFB-HG 26.5 ± 0.9, and for the marketed formulation 29.5 ± 0.3 mm. The present study data shown in Table 6, and Fig. 6, exhibited an acceptable range of ZOI, which is comparable with earlier reports.
Table 6.
Anti-bacterial activity analysis.
| Bacteria | ZOI (mm) |
||
|---|---|---|---|
| Standard (Doxycycline) | N-HG | N-SFB-HG | |
| Gram + ve & Gram-ve | 29.5 ± 0.3 | 26 ± 0.7 | 26.5 ± 0.9 |
*Anti-bacterial study was performed in triplicates (n = 3) and outcomes expressed as mean ± SD.
Fig. 6.
In vitro anti-bacterial study showing ZOI.
3.10. Skin irritation study
To examine irritation, N-HG, N-SFB-HG, and base gel were evenly applied to the shaved area (2 cm2). Each animal was evaluated individually for any type of reaction at the site of application and recorded for every 1, 24, 48, and 72 h, on 7th, and 10th days. No allergy, swelling, or erythema was observed for developed N-HG and N-SFB-HGs and base gel formulation (Fig. 7).
Fig. 7.
Rat’s dorsal area shaving and application of hydrogels.
3.11. Ex vivo permeation study
Ex vivo gel penetration in rat skin is shown in Fig. 7. A total of 6.9, 9.4, and 13.5 μg/ cm2 of neem permeated through the neem extract solutions N-HG and N-SFB-HG, respectively. It was revealed that the capacity of neem permeation was significantly enhanced in SFB containing hydrogels.
3.12. In vivo wound healing activity studies
The wound for each group of animals from days 0 to 21 is shown in Fig. 9 (group 1: Control/base HG treated, group 2: N-HG treated, and group 3: N-SFB-HG treated). The recovery rate of wounds in animals, when compared with all other groups, the N-SFB-HG-treated group demonstrated a rapid regeneration of the tissues and a substantial difference in wound closure. The wound shrinkage percentage for various groups on days 7, 14, and 21 is shown in Fig. 9. The N-SFB-HG treated group showed the most significant wound closure rates during the days listed above (53.8, 73.5, and 99.12%, respectively). Compared to the N-SFB-HG treated group, the N-HG treated group demonstrated wound contraction of 36.66, 48.88, and 79.25%, correspondingly. The control group also showed wound contraction on the corresponding days of 9.73, 19.73, and 27.83 %. Curiously, on the 18th day, the rats treated with N-SFB-HG had 98.99 % wound contraction can be observed clearly in Fig. 9. When compared to the N-HG and base gel treated groups, a remarkable difference in the percentage of contraction wounds was seen for N-SFB-HG throughout the trial (p < 0.0001).(See Fig. 10).
Fig. 9.
Shows wound healing activity of formulated HGs in experimental rats using an excision wound model.
Fig. 10.
Wound contraction (%)of N-SFB-HG, N-HG treated, and control-treated rats on 7th, 14th, and 21st day. * Signifies a significant difference in the contraction of the wound. ****=p < 0.0001, ***=p < 0.001.
3.13. Transmission electron microscopy (TEM) analysis
TEM images of N-HG and N-SFB-HG are depicted in Fig. 11. The outcomes clearly showed that both the hydrogels' morphology has a homogenous nature, little porosity, and merely aggregative properties.
Fig. 11.
TEM images of (A) N-HG and (B)N-SFB-HG.
3.14. Stability study
As per the ICH guidelines for prepared N-HG and N-SFB-HG formulations, a stability study was conducted to confirm safety and efficacy during storage. The formulations were found to have stable quality attributes over 3 months. The findings of the stability studies confirmed that the formulated HGs were stable, as the data shown in Table 7.
Table 7.
Accelerated stability study analysis of neem hydrogels.
| Physical Parameters | At 25 ± 2℃ Temperature |
At 40 ± 2℃ Temperature |
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 30th Day |
60th day |
90th day |
30th Day |
60th day |
90th day |
|||||||
| N-HG | N-SFB-HG | N-HG | N-SFB-HG | N-HG | N-SFB-HG | N-HG | N-SFB-HG | N-HG | N-SFB-HG | N-HG | N-SFB-HG | |
| Visual appearance | Thick & smeared throughout the study | |||||||||||
| Spreadability (mm/min) | 25 ± 1.1 | 23 ± 2.7 | 24 ± 1.6 | 25 ± 1.1 | 23 ± 2.7 | 24 ± 1.6 | 24 ± 2.1 | 24 ± 1.7 | 23 ± 2.6 | 24 ± 2.1 | 22 ± 1.3 | 25 ± 2.2 |
| pH | 5.9 ± 0.1 | 5.8 ± 0.7 | 5.7 ± 0.8 | 5.9 ± 0.1 | 5.8 ± 0.7 | 5.7 ± 0.8 | 5.8 ± 0.6 | 5.7 ± 0.9 | 5.8 ± 0.5 | 5.9 ± 0.2 | 5.8 ± 0.2 | 5.8 ± 0.4 |
| Viscosity (cPs) | 54 ± 1.5 | 55 ± 3.9 | 55 ± 1.8 | 54 ± 1.5 | 55 ± 3.9 | 55 ± 1.8 | 58 ± 2.5 | 57 ± 2.9 | 56 ± 2.8 | 55 ± 2.5 | 56 ± 2.9 | 58 ± 2.4 |
| Drug content (%) | 83 ± 2.11 | 83 ± 3.21 | 84 ± 1.12 | 86 ± 2.21 | 88 ± 1.16 | 83 ± 2.21 | 84 ± 3.14 | 86 ± 4.10 | 85 ± 2.14 | 86 ± 3.21 | 86 ± 2.69 | 87 ± 1.05 |
*All the analysis were carried out in triplicates (n = 3) and outcomes are expressed as mean ± SD.
4. Discussion
The current work aimed to prepare Neem Extract Hydrogels (N-HG) and Neem Silk Fibroin hydrogel (N-SFB-HG) and evaluate the prospective of combinational polymeric materials in skin regeneration. Qualitative phytochemical analysis based on methods proposed earlier several research (Muhammad and Abubakar, 2016, Uwague, 2019, Khanal, 2021) revealed the presence of alkaloids, flavonoids, saponins, terpenoids, polyphenols, and tannins. In contrast to their findings, our study also indicated that the neem leaf extract contained glycosides. Our research confirmed the absence of coumarins and matched the findings (Fig. 4) as reported by Khanal, 2021.Biuet al. (2009) reported that aqueous neem leaf extract contains more saponins and fewer alkaloids, which correlates with our result. Earlier studies demonstrated that SFB has the potential for tissue engineering and regeneration (Chen and Liu, 2016, Gil et al., 2013).The findings of the IR spectrum analysis showed (Fig. 5 and Table 3) that the hydrogels and the excipients incorporated in the formulation were compatible. The data chart in Table 4 shows how the developed N-HG and N-SFB-HGs were uniform in texture and had good spreadability(Pawar et al., 2017). The spreadability outcomes of gels in this range showed that using a low shear rate made them easier to spread over areas of skin, and several studies supported this observation (Chen and Liu, 2016). The drug content (Table 4) of the present study findings was comparable with the earlier results reported by Raju and Jose, (2019). Neither bacterial nor fungal growth was found during the sterility test (Table 5).
A good zone of inhibition supported the developed formulation's anti-bacterial activity. However, as shown in Table 6 and Fig. 6, adding SFB did not considerably enhance that anti-bacterial effect. Similar findings were made by other researchers, who discovered that while SFB alone has no anti-bacterial effect, antibiotics can be delivered continuously from SFB biomaterials to stop the growth of bacteria (El-Kased, 2016). It demonstrated the sterility of the testing media, N-HG, and N-SFB-HG. Hence, the sterility test was passed by the N-HG and N-SFB-HG formulations. Similar results were reported from a study conducted by Narayana et al., 2023. Both N-HG and N-SFB-HGs and gel base formulations exhibited no erythema, edema, or allergies upon application on rats (Fig. 7). The results of the current study are comparable to those of past studies like Babu et al., 2019. The permeation capacity of neem was also much higher (Fig. 8) in N-SFB-HG than in N-HG and plain neem extract solution, which may be linked to the porous interconnected structure of N-SFB-HG (Dubey and Prabhu, 2014). Fig. 9,10 shows results from in vivo tests on animals showing that animals treated with N-SFB-HG had faster wound healing. On day 17, N-SFB-HG saw a total wound recovery (99.50%). This could be a result of the greater concentration of β-sheet patterns in SF, which tremendously encourages adhesion, proliferation, and migration of cells, hence promoting tissue regeneration around the injured area. The adhesion capabilities of the gel were improved by the porous interconnecting well-distributed molecules in N-SFB-HG, according to the HR-TEM findings shown in Fig. 11 (Zhang et al., 2009). According to already published research, SF’s porous and linked structure may promote speedier wound healing and tissue regeneration (He et al., 2019).
Fig. 8.
Ex vivo permeation study of Neem extract, N-HG, and N-SFB-HG.
The HGs' shelf life, as indicated in Table 7, demonstrates no change in viscosity, pH, spreadability, color, odor, drug content, and effect of centrifugation. The findings confirmed the desired stability of the designed HGs. Excellent statistics, a sound design, and encouraging findings from the current study indicate a path for advancements in wound healing therapies. The limited in vivo study sample size and absence of histological evaluation data for wound tissues may be limitations of the study.
5. Conclusions
This study designed and assessed several properties of neem hydrogel formulations alone and combined with SF. The N-HG and N-SFB-HG proved exceptional biocompatibility, desirable stability, and satisfactory physical features. In an in vivo animal model, N-SFB-HG significantly decreased the time duration needed for tissue reconstruction compared to control and N-HG-treated animal groups. Even though many research groups already work with neem and SF for wound healing effects, the current novel combination treatment approach demands attention for effective healing therapeutics. This implies that the N-SFB-HG might be used in the long term to design novel wound-healing therapeutics to ensure conformance for both patients and the medical industry.
Funding
The authors are thankful to AlMaarefa University for supporting this research.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
The authors would like to acknowledge the Researchers Supporting Project number (RSP2023R115), King Saud University, Riyadh, Saudi Arabia, for extending financial support to do this research project. Researchers acknowledge Sericare, Bangalore, for supplying the gift sample of silk fibroin used in the investigation. The Yenepoya Pharmacy College and Research Center provided the laboratory space used for the authors' experimental work, for which they are grateful.
Footnotes
Peer review under responsibility of King Saud University.
Contributor Information
Arfa Nasrine, Email: arfanasrine14@gmail.com.
Soumya Narayana, Email: kalikollur123@gmail.com.
Mohammed Gulzar Ahmed, Email: mohammedgulzar1@gmail.com.
Rokeya Sultana, Email: rokeya009ster@gmail.com.
Mansour Almuqbil, Email: mmetwazi@ksu.edu.sa.
Moneer E. Almadani, Email: mmadani@mcst.edu.sa.
Ahmed Alshehri, Email: adalshehri@iau.edu.sa.
Adel Alghamdi, Email: Ai.alghamdi@bu.edu.sa.
Sultan Alshehri, Email: sshehri.c@mcst.edu.sa.
Syed Mohammed Basheeruddin Asdaq, Email: sasdaq@gmail.com, sasdag@mcst.edu.sa.
References
- Ahamed M.I., Sastry T.P. Wound dressing application of chitosan-based bioactive compounds. Int. J. Pharm. Life Sci. 2011 Aug 1;2(8) [Google Scholar]
- Aiyalu R., Govindarjan A., Ramasamy A. Formulation and evaluation of topical herbal gel for the treatment of arthritis in animal model. Braz. J. Pharm. Sci. 2016 Sep;52(3):493–507. [Google Scholar]
- Al-Hashemi Z.S., Hossain M.A. Biological activities of different neem leaf crude extracts used locally in Ayurvedic medicine. Pac. Sci. Rev. A: Nat. Sci. Eng. 2016 Jul 1;18(2):128–131. [Google Scholar]
- Al-Suwayeh S.A., Taha E.I., Al-Qahtani F.M., Ahmed M.O., Badran M.M. Evaluation of skin permeation and analgesic activity effects of carbopollornoxicam topical gels containing penetration enhancer. Scient. World J. 2014 Jan;1:2014. doi: 10.1155/2014/127495. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Altman G.H., Diaz F., Jakuba C., Calabro T., Horan R.L., Chen J., Lu H., Richmond J., Kaplan D.L. Silk-based biomaterials. Biomaterials. 2003 Feb 1;24(3):401–416. doi: 10.1016/s0142-9612(02)00353-8. [DOI] [PubMed] [Google Scholar]
- Anbarasan B., Kumar R.S., Thanka J., Ramaprabhu S., Shanmuganathan S. Preparation and characterization of pH based carbopol 934p in-situ hydrogels for the treatment of harmful bacterial infections. Int. J. Pharm. Sci. Res. 2019 Jan 1;10(1):232–244. [Google Scholar]
- Aslani A, Zolfaghari B, Fereidani Y. Design, formulation, and evaluation of a herbal gel contains melissa, sumac, licorice, rosemary, and geranium for treatment of recurrent labial herpes infections. Dent Res J (Isfahan). 2018 May-Jun;15(3):191-200. doi: 10.4103/1735-3327.231865. PMID: 29922338; PMCID: PMC5958536. [DOI] [PMC free article] [PubMed]
- Babu C.A., Rao P., Anitha N., Latha S.B., Anusha C.L., Parishudda K., Pragathi S. Design and characterization of hydrogel formulations containing aloe vera and neem seed oil. J. Med. Pharm. Allied Sci. 2019;8(4):2329–2341. [Google Scholar]
- Bhowmik D, Gopinath H, Kumar BP, Duraivel S, Kumar KS. Recent advances in novel topical drug delivery system. The Pharma Innovation. 2012 Nov 1;1(9, Part A):12.
- Biu A.A., Yusufu S.D., Rabo J.S. Phytochemical screening of Azadirachtaindica (Neem)(Meliaceae) in Maiduguri, Nigeria. Biosci. Res. Commun. 2009;21(6):281–283. [Google Scholar]
- Chen M.X., Alexander K.S., Baki G. Formulation and evaluation of anti-bacterial creams and gels containing metal ions for topical application. J. Pharm. 2016;2016 doi: 10.1155/2016/5754349. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen F.M., Liu X. Advancing biomaterials of human origin for tissue engineering. Prog. Polym. Sci. 2016 Feb;1(53):86–168. doi: 10.1016/j.progpolymsci.2015.02.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chittodiya P., Tomar R.S., Ramchandani U., Manocha N., Agrawal S. Topical gel-A review. Int. J. Pharma. Biol. Arch. 2013;4(4):606–613. [Google Scholar]
- Chundran N.K., Husen I.R., Rubianti I. Effect of Neem leaves extract (AzadirachtaIndica) on wound healing. Althea Med. J. 2015 Jun 30;2(2):199–203. [Google Scholar]
- Diegelmann R.F., Evans M.C. Wound healing: an overview of acute, fibrotic and delayed healing. Front. Biosci. 2004;9:283–289. doi: 10.2741/1184. [DOI] [PubMed] [Google Scholar]
- Dubey A., Prabhu P. Formulation and evaluation of stimuli-sensitive hydrogels of timolol maleate and brimonidine tartrate for the treatment of glaucoma. Int. J. Pharma. Investig. 2014 Jul;4(3):112. doi: 10.4103/2230-973X.138340. [DOI] [PMC free article] [PubMed] [Google Scholar]
- El-Kased R.F. Natural antibacterial remedy for respiratory tract infections. Asian Pac. J. Trop. Biomed. 2016 Mar 1;6(3):270–274. [Google Scholar]
- Fong Yen W., Basri M., Ahmad M., Ismail M. Formulation and evaluation of galantamine gel as drug reservoir in transdermal patch delivery system. Scient. World J. 2015;2015 doi: 10.1155/2015/495271. Epub 2015 Feb 26. PMID: 25853145; PMCID: PMC4374332. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gil E.S., Panilaitis B., Bellas E., Kaplan D.L. Functionalized silk biomaterials for wound healing. Adv. Healthc. Mater. 2013;2(1):206–217. doi: 10.1002/adhm.201200192. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gonzalez A.C., Costa T.F., Andrade Z.D., Medrado A.R. Wound healing-A literature review. An. Bras. Dermatol. 2016 Oct;91(5):614–620. doi: 10.1590/abd1806-4841.20164741. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gowda B.H., Mohanto S., Singh A., Bhunia A., Abdelgawad M.A., Ghosh S., Ansari M.J., Pramanik S. Nanoparticle-based therapeutic approaches for wound healing: a review of the state-of-the-art. Mater. Today Chem. 2023 Jan;1(27) [Google Scholar]
- Harborne J.B. Phytochemical Methods. Springer; Dordrecht: 1973. Phenolic compounds; pp. 33–88. [DOI] [Google Scholar]
- He S., Shi D., Han Z., et al. Heparinized silk fibroin hydrogels loading FGF1 promote the wound healing in rats with full-thickness skin excision. Bio. Med. Eng. OnLine. 2019;18:97. doi: 10.1186/s12938-019-0716-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hees C.T. Understanding the barriers to healing. Adv. Skin Wound Care. 2012;25(5):240. doi: 10.1097/01.ASW.0000414709.35053.f9. [DOI] [PubMed] [Google Scholar]
- Khanal S. Qualitative and Quantitative Phytochemical Screening of AzadirachtaindicaJuss. Plant Parts. International Journal of Applied Sciences and Biotechnology. 2021 Jun 28;9(2):122-7.3(4): 501-05.
- Kumar V, Abbas AK, Fausto N, Aster JC. Patologia: bases patológicas das doenças. Rio de Janeiro. 2005.
- Kumar N., Saxena G., Shukla S., Seliya M., Samuel A. Formulation and evaluation of contraceptive herbal gel. Pharma Innov. J. 2018;7(11):520–523. [Google Scholar]
- Lu S., Li J., Zhang S., Yin Z., Xing T., Kaplan D.L. The influence of the hydrophilic–lipophilic environment on the structure of silk fibroin protein. J. Mater. Chem. B. 2015;3(13):2599–2606. doi: 10.1039/c4tb01873g. [DOI] [PubMed] [Google Scholar]
- MacKay D., Miller A.L. Nutritional support for wound healing. Altern. Med. Rev. 2003;8(4):359–377. [PubMed] [Google Scholar]
- Mandal S., Thimmasetty M.K., Prabhushankar G.L., Geetha M.S. Formulation and evaluation of an in situ gel-forming ophthalmic formulation of moxifloxacin hydrochloride. Int. J. Pharm. Investig. 2012;2(2):78–82. doi: 10.4103/2230-973X.100042. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McDonald A., Hoffman M., Hedner U., Roberts H.R., Monroe D.M. Restoring hemostatic thrombin generation at the time of cutaneous wounding does not normalize healing in hemophilia B. J. Thromb. Haemost. 2007 Aug;5(8):1577–1583. doi: 10.1111/j.1538-7836.2007.02647.x. [DOI] [PubMed] [Google Scholar]
- Minoura N., Tsukada M., Nagura M. Fine structure and oxygen permeability of silk fibroin membrane treated with methanol. Polymer. 1990 Feb 1;31(2):265–269. [Google Scholar]
- Misal G., Dixit G., Gulkari V. Formulation and evaluation of herbal gel. Indian J. Nat. Prod. Resour. 2012;3(4):501–505. [Google Scholar]
- Monica A.S., Gautami J. Design and evaluation of topical hydrogel formulation of diclofenac sodium for improved therapy. Int. J. Pharm. Sci. Res. 2014 May;1(5):1973–1980. [Google Scholar]
- Muhammad S.A., Abubakar S.M. Qualitative and quantitative determination of phytochemicals in aqueous extract of chrysophyllumalbidum seed kernel. Biosci. Biotechnol. Res. Asia. 2016 Jun 25;13(2):1201–1206. [Google Scholar]
- Narayana S., Ahmed M.G. Design and evaluation of ocular hydrogel containing combination of ofloxacin and dexamethasone for the treatment of conjunctivitis. Braz. J. Pharm. Sci. 2022 Nov;25:58. [Google Scholar]
- Narayana S., Nasrine A., Ahmed M.G., Sultana R., Gowda B.J., Surya S., et al. Potential benefits of using chitosan and silk fibroin topical hydrogel for managing wound healing and coagulation. Saudi Pharma. J. 2023 Feb 1 doi: 10.1016/j.jsps.2023.01.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nasrine A., Ahmed M.G., Narayana S. Silk fibroin-anastrozole loaded prolonged-release biodegradable nanomedicine: A promising drug delivery system for breast cancer therapy. Mater. Today:. Proc. 2022 Jan;1(68):56–65. [Google Scholar]
- Okamoto Y., Yano R., Miyatake K., Tomohiro I., Shigemasa Y., Minami S. Effects of chitin and chitosan on blood coagulation. Carbohydr. Polym. 2003 Aug 15;53(3):337–342. [Google Scholar]
- Padol AR, Jayakumar K, Shridhar NB, Mohan K, Manochaya S. PRECLINICAL SAFETY ASSESSMENT OF SILK PROTEIN DERIVED BIOMATERIAL IN LABORATORY ANIMALS. Journal of Cell and Tissue Research. 2013 May 1;13(2):0000-.
- Pawar J., Narkhede R., Amin P., Tawde V. Design and evaluation of topical diclofenac sodium gel using hot melt extrusion technology as a continuous manufacturing process with Kolliphor® P407. AAPS PharmSciTech. 2017 Aug;18(6):2303–2315. doi: 10.1208/s12249-017-0713-5. [DOI] [PubMed] [Google Scholar]
- Polaquini SR, Svidzinski TI, Kemmelmeier C, Gasparetto A. Effect of aqueous extract from Neem (Azadirachtaindica A. Juss) on hydrophobicity, biofilm formation and adhesion in composite resin by Candida albicans. Archives of oral biology. 2006 Jun 1;51(6):482-90. [DOI] [PubMed]
- Raju D., Jose J. Development and evaluation of novel topical gel of neem extract for the treatment of bacterial infections. J. Cosmet. Dermatol. 2019 Dec;18(6):1776–1783. doi: 10.1111/jocd.12965. [DOI] [PubMed] [Google Scholar]
- Sanjana A, Ahmed MG, Gowda BH, Surya S. Formulation and characteristic evaluation of tacrolimus cubosomal gel for vitiligo. JOURNAL OF DISPERSION SCIENCE AND TECHNOLOGY. 2022 Oct 25.
- Santin M., Motta A., Freddi G., Cannas M. In vitro evaluation of the inflammatory potential of the silk fibroin. J. Biomed. Mater. Res.: Off. J. Soc. Biomater., Japanese Soc. Biomater., Australian Soc. Biomater. Korean Soc. Biomater. 1999 Sep 5;46(3):382–389. doi: 10.1002/(sici)1097-4636(19990905)46:3<382::aid-jbm11>3.0.co;2-r. [DOI] [PubMed] [Google Scholar]
- Septiyani R, Wibowo C. Identification of active compounds and testing the antioxidant properties of neem leaf extract. InAIP Conference Proceedings 2019 Apr 17 (Vol. 2094, No. 1, p. 020034). AIP Publishing LLC.
- Shaw T.J., Martin P. Wound repair at a glance. J. Cell Sci. 2009 Sep 15;122(18):3209–3213. doi: 10.1242/jcs.031187. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shrestha P., Adhikari S., Lamichhane B., Shrestha B.G. Phytochemical screening of the medicinal plants of Nepal. IOSR J. Environ. Sci. Toxicol. Food Technol. 2015;6:11–17. [Google Scholar]
- Singer AJ, Clark RA. Cutaneous wound healing. N Eng J Med. 1999;34(10):738–46. 6. [DOI] [PubMed]
- Singh V., Bushetti S.S., Appala R., Shareef A., Imam S.S., Singh M. Stimuli-sensitive hydrogels: A novel ophthalmic drug delivery system. Indian J. Ophthalmol. 2010 Nov;58(6):477. doi: 10.4103/0301-4738.71677. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sixma J.J., Van Den Berg A. The haemostaticplug inhaemophilia A: a morphological study of haemostatic plug formation in bleeding time skin wounds of patients with severe haemophilia A. Br. J. Haematol. 1984 Dec;58(4):741–753. doi: 10.1111/j.1365-2141.1984.tb06121.x. [DOI] [PubMed] [Google Scholar]
- Subapriya R., Nagini S. Medicinal properties of neem leaves: a review. Curr. Med. Chem. Anticancer Agents. 2005;5(2):149–156. doi: 10.2174/1568011053174828. [DOI] [PubMed] [Google Scholar]
- Tas C., Ozkan Y., Savaşer A., Baykara T. In vitro and ex vivo permeation studies of chlorpheniramine maleate gels prepared by carbomer derivatives. Drug Dev. Ind. Pharm. 2004 Jan 1;30(6):637–647. doi: 10.1081/ddc-120037665. [DOI] [PubMed] [Google Scholar]
- Uwague A. Nigeria; Delta State: 2019. Comparative potential qualitative and quantitative phytochemical evaluation of neem and Moringa Oleifera leaf plants in Ozoro; pp. 120–340. [Google Scholar]
- Zhang Q., Yan S., Li M. Silk fibroin based porous materials. Materials. 2009 Dec 9;2(4):2276–2295. [Google Scholar]